nci-pbcf-ccl247 (hct 116) colorectal carcinoma … · nci-pbcf-ccl247 (hct 116) colorectal...

NCI-PBCF-CCL247 (HCT 116) Colorectal Carcinoma

(ATCCregCCL-247

trade )

February 27 2012 Version 16

Table of Contents

1 BACKGROUND INFORMATION ON HCT 116 CELL LINE 3

2 GENERAL INFORMATION FOR THE THAWING PROPAGATING AND CRYOPRESERVING OF NCI-PBCF- CCL247 (HCT 116) 3

3 REAGENTS 5

A PREPARATION OF COMPLETE GROWTH MEDIUM (MCCOYrsquoS 5A + 10 (VV) FBS) 5

4 THAWING AND PROPAGATION OF CELLS 6

A THAWING CELLS 6 B PROPAGATING CELLS 7 C SUBCULTURING CELLS 8

5 HARVESTING OF CELLS FOR CRYOPRESERVATION 9

6 CRYOPRESERVATION OF CELLS 11

A CRYOPRESERVATION USING A RATE-CONTROLLED PROGRAMMABLE FREEZER 11 i Using the Cryomed 11

B CRYOPRESERVATION USING ldquoMR FROSTYrdquo 12

7 STORAGE 13

APPENDIX 1 PHOTOMICROGRAPHS OF NCI-PBCF-CCL247 (HCT 116) CELLS 14

APPENDIX 2 GROWTH PROFILE OF NCI-PBCF-CCL247 (HCT 116) CELLS 16

APPENDIX 3 CYTOGENETIC ANALYSIS OF NCI-PBCF-CCL247 HCT 116 CELLS 17

APPENDIX 4 GLOSSARY OF TERMS 20

APPENDIX 5 REFERENCE 21

APPENDIX 6 REAGENT LOT TRACEABILITY AND CELL EXPANSION TABLES 22

APPENDIX 7 CALCULATION OF POPULATION DOUBLING LEVEL (PDL) 24

APPENDIX 8 SAFETY PRECAUTIONS 24

SOP Thawing Propagation and Cryopreservation of NCI-PBCF-CCL247 (HCT 116)

Protocol for Thawing Propagation and Cryopreservation of NCI-PBCF-CCL247 (HCT 116) (ATCCregCCL-247 trade )

colorectal carcinoma

1 Background Information on HCT 116 cell line

Designations HCT 116

Biosafety Level 1

Shipped frozen (in dry ice)

Growth Properties adherent (See Appendix 1)

Organism Homo sapiens

Source Organ colon

Disease colorectal carcinoma

For more information visit the ATCC webpage

httpwwwatccorgATCCAdvancedCatalogSearchProductDetailstabid452DefaultaspxA

TCCNum=CCL-247ampTemplate=cellBiology

2 General Information for the thawing propagating and

cryopreserving of NCI-PBCF- CCL247 (HCT 116)

Culture Initiation

The cryoprotectant (DMSO) should be removed by centrifugation

The seeding density to use with a vial of HCT 116 cells is about 10 x 105

cellscm2

or one vial into a T-25 flask containing 10 mL of complete growth

medium is (McCoyrsquos 5a + 10 (vv) FBS)

Complete growth

medium

The complete growth medium used to expand HCT 116 cells is McCoyrsquos 5a + 10

(vv) FBS

Complete growth medium (McCoyrsquos 5a + 10 (vv) FBS) should be pre-warmed

before use by placing into a water bath set at 35 oC to 37

oC for 15 min to 30 min

After 30 min the complete growth medium (McCoyrsquos 5a + 10 (vv) FBS) should

be moved to room temperature until used Complete growth medium McCoyrsquos 5a + 10 (vv) FBS) should be stored at 2

oC to 8

oC when not in use

Cell Growth

The growth temperature for HCT 116 is 37 oC plusmn 1

oC

A 5 + 1 CO2 in air atmosphere is recommended

Growth Properties Population doubling time (PDT) is approximately 21 h (see Figure 4)

Special Growth

Requirements

Subculture HCT 116 cells at 80 to 90 confluence or when cell density

reaches an average of 4 x 105 cellscm

2

Page 3 of 24


Subculture Medium

025 (wv) trypsin-053 mM EDTA (ATCC cat no 30-2101)

Subculturing reagents should be pre-warmed before use by placing into a water

bath set at 35 oC to 37


After 30 min the subculturing medium should be moved to room temperature

until used Subculturing reagents should be stored at 2 oC to 8

oC when not in

use

Subculture Method

The attached HCT 116 cells are subcultured using 025 (wv) trypsin-053 mM

EDTA (ATCC cat no 30-2101)

The enzymatic action of the trypsin-EDTA is stopped by adding complete growth

medium to the detached cells

A split ratio of 18 to 110 or a seeding density of 4 x 104

viable cellscm 2

to5 x 104

viable cellscm 2

is used when subculturing HCT 116 cells

Viable

CellsmLCryovial

The target number of viable cellsmLcryovial is 3 x 106

(acceptable range 20 x

106

viable cellsmL to 30 x 106

viable cellsmL)

Cryopreservation

Medium

The cryopreservation medium for HCT 116 cells is complete growth medium

(McCoyrsquos 5a + 10 (vv) FBS) containing 5 (vv) DMSO (ATCC cat no 4-X)

General Procedure to be applied throughout the SOP

Use of good aseptic technique is critical Any materials that are contaminated as

well as any materials with which they may have come into contact must be

disposed of immediately Aseptic Technique

Traceability of

materialreagents

Record the manufacturer catalog number lot number date received date

expired and any other pertinent information for all materials and reagents used

Record information in the Reagent Lot Traceability Table 4 (Appendix 6)

Record the subculture and growth expansion activities such as passage number

confluence viability cell morphology (see Figures 1-3 Appendix 1) and

population doubling levels (PDLs) in the table for Cell Expansion (Table 5

Appendix 6) Calculate PDLs using the equation in Appendix 7

Expansion of cell line

Medium volumes Medium volumes are based on the flask size as outlined in Table 1

Glossary of Terms

Safety Precaution

Refer to Glossary of Terms used throughout the document (see Appendix 4)

Refer to Safety Precautions pertaining to the thawing propagating and

cryopreserving of HCT 116 (see Appendix 8)

Page 4 of 24


Table 1 Medium Volumes

Flask Size Medium Volume Range

125 cm2

(T-125) 3 mL to 6 mL

25 cm2

(T-25) 5 mL to 13 mL

75 cm2

(T-75) 10 mL to 38 mL

150 cm2

(T-150) 30 mL to 75 mL

175 cm2

(T-175) 35 mL to 88 mL

225 cm2

(T-225) 45 mL to 113 mL

3 Reagents

Follow Product Information Sheet storage andor thawing instructions Below is a list of

reagents for the propagation subcultivation and cryopreservation of HCT 116 cells

Table 2 Reagents for Expansion Subculturing and Cryopreservation of HCT 116 cells

Complete growth medium reagents

Subculturing reagents Cryopreservation medium reagents

McCoyrsquos 5a Medium Modified (ATCC cat no 30-2007)

Trypsin-EDTA (025 (wv)

Trypsin053 mM EDTA )

(ATCC cat no 30-2101)


10 (vv) Fetal Bovine

Serum (FBS)


Dulbeccorsquos Phosphate Buffered Saline (DPBS) modified without

calcium chloride and without

magnesium chloride


10 (vv) FBS (ATCC cat no 30-2020)

5 (vv) Dimethyl Sulfoxide (DMSO) (ATCC cat no 4-X)

a Preparation of complete growth medium (McCoyrsquos 5a + 10

(vv) FBS)

The complete growth medium is prepared by aseptically combining

1 56 mL FBS (ATCC cat no 30-2020) to a 500 mL bottle of basal medium ndash McCoyrsquos 5a (ATCC cat no 30-2007)

2 Mix gently by swirling

Page 5 of 24


4 Thawing and Propagation of Cells

Reagents and Material

Complete growth medium (McCoyrsquos 5a + 10 (vv) FBS)

Water bath

T-25 cm2 polystyrene flask

15 mL polypropylene conical centrifuge tubes

Plastic pipettes (1 mL10 mL 25 mL)

a Thawing cells

Method

1 Place complete growth medium (McCoyrsquos 5a + 10 (vv) FBS) in a water bath set at

35 oC to 37 oC

2 Label T-25 flask to be used with the

(a) name of cell line (b) passage number (c) date (d) initials of technician

3 Wearing a full face shield retrieve a vial of frozen cells from liquid nitrogen freezer

4 Thaw the vial by gentle agitation in a water bath set at 35 oC to 37 oC To reduce the

possibility of contamination keep the O-ring and cap out of the water

5 Note Thawing should be rapid (approximately 2 min to 3 min just long enough

for most of the ice to melt)

6 Remove vial from the water bath and process immediately

7 Remove excess water from the vial by wiping with sterile gauze saturated with 70

ethanol

8 Transfer the vial to a BSL-2 laminar-flow hood

Page 6 of 24


b Propagating cells

Method

1 Add 9 mL of complete growth medium (McCoyrsquos 5a + 10 (vv) FBS) to a 15-mL conical centrifuge tube

2 Again wipe the outer surface of the vial with sterile gauze wetted with 70 ethanol

3 Using sterile gauze carefully remove the cap from the vial

4 With a 1 mL pipette transfer slowly the completely thawed content of the vial (1 mL cell suspension) to the 15-mL conical centrifuge tube containing 9 mL complete growth medium (McCoyrsquos 5a + 10 (vv) FBS) Gently resuspend cells by pipetting up and down

5 Centrifuge at 125 xg at room temperature for 8 min to 10 min

6 Carefully aspirate (discard) the medium leaving the pellet undisturbed

7 Using a 10 mL pipette add 10 mL of complete growth medium (McCoyrsquos 5a + 10 (vv) FBS)

8 Resuspend pellet by gentle pipetting up and down

9 Using a 1 mL pipette remove 1 mL of cell suspension for cell count and viability Cell counts are performed using either an automated counter (such as Innovatis Cedex System Beckman-Coulter ViCell system) or a hemocytometer

10 Record total cell count and viability When an automated system is used attach copies of the printed results to the record

11 Plate cells in pre-labeled T-25 cm2 flask at about 10 x 105 cellscm2

12 Transfer flask to a 37 degCplusmn 1degC in 5 CO2 incubator if using flasks with vented caps (for non-vented caps stream 5 CO2 in the headspace of flask)

13 Observe culture daily by eye and under an inverted microscope to ensure culture is free of contamination and culture has not reached confluence Monitor visually the pH of the medium daily If the medium goes from red through orange to yellow change the medium

14 Note In most cases cultures at a high cell density exhaust the medium faster than those at low cell density as is evident from the change in pH A drop in pH is usually accompanied by an increase in cell density which is an indicator to subculture the cells Cells may stop growing when the pH is between pH 7 to pH 6 and loose viability between pH 65 and pH 6

15 If fluid renewal is needed aseptically aspirate the complete growth medium from the flask and discard Add an equivalent volume of fresh complete growth medium to the flask Alternatively perform a fluid addition by adding fresh complete growth medium to the flask without removing the existing medium Record fluid change or fluid addition on the Cell Line Expansion Table (see Table 5 in Appendix 6)

Page 7 of 24


16 If subculturing of cells is needed continue to lsquoSubculturing cellsrsquo

Note Subculture when cells are 80-90 confluent (see photomicrographs in Appendix 1)

c Subculturing cells


025 (wv) Trypsin-053 mM EDTA

DPBS

Complete growth medium (McCoyrsquos 5a (ATCC cat no 30-2007) + 10 (vv) FBS


Plastic pipettes (1 mL 10 mL 25 mL)

T-75 cm2 T-225 cm2 polystyrene flasks

Method

1 Aseptically remove medium from the flask

2 Add appropriate volumes of sterile Ca2+- and Mg2+-free DPBS to the side of the flask opposite the cells so as to avoid dislodging the cells (see Table 3)

3 Rinse the cells with DPBS (using a gently rocking motion) and discard

4 Add appropriate volume of 025 (wv) Trypsin-053 mM EDTA solution to the flask (see Table 3)

5 Incubate the flask at 37 oC plusmn 1 oC until the cells round up Observe cells under an inverted microscope every 5 min When the flask is tilted the attached cells should slide down the surface This usually occurs after 5 min to 10 min of incubation

Note Do not leave trypsin-EDTA on the cells any longer than necessary as clumping may result

6 Neutralize the trypsin-EDTAcell suspension by adding an equal volume of complete growth medium (McCoyrsquos 5a + 10 (vv) FBS) to each flask Disperse the cells by pipetting gently over the surface of the monolayer Pipette the cell suspension up and down with the tip of the pipette resting on the bottom corner or edge until a single cell suspension is obtained Care should be taken to avoid the creation of foam

7 Using a 1 mL pipette remove 1 mL of cell suspension for total cell count and viability

8 Record total cell count and viability

9 Add appropriate volume of fresh complete growth medium (McCoyrsquos 5a + 10 (vv) FBS) and transfer cell suspension (for volume see Table 1 on page 4) into new pre-labeled flasks at a seeding density of 4 x 104 viable cellscm2 to 5 x 104 viable cellscm2 or a split ratio of 18 to 110

Page 8 of 24

DPBS Rinse

Flask Type Flask Size Buffer Trypsin-EDTA

125 cm2

(T-125) 1 mL to 3 mL 1 mL to 2 mL T-flask

25 cm2

(T-25) 1 mL to 5 mL 1 mL to 3 mL

75 cm2

(T-75) 4 mL to 15 mL 2 mL to 8 mL

150 cm2

(T-150) 8 mL to 30 mL 4 mL to 15 mL

175 cm2

(T-175) 9 mL to 35 mL 5 mL to 20 mL

225 cm2

(T-225) 10 mL to 45 mL 5 mL to 25 mL


10 Label all new flasks with the


Table 3 - Volume of Rinse Buffer and Trypsin

5 Harvesting of Cells for Cryopreservation



DPBS


(ATCC cat no 30-2020))

50 mL or 250 mL conical centrifuge tube


Sterile DMSO

1 mL to 18 mL cryovials

Ice bucket with ice

Page 9 of 24


Method

1 Label cryovials to include information on the (a) name of cell line (b) passage

number (c) date

2 Prepare cryopreservation medium by adding DMSO to cold complete growth medium

(McCoyrsquos 5a + 10 (vv) FBS) at a final concentration of 5 (vv) DMSO Place

cryopreservation medium on ice until ready to use


4 Add appropriate volumes of sterile Ca2+- and Mg2+-free DPBS to the side of the flask so as to avoid dislodging the cells (see Table 3)








11 Spin cells at approximately 125 xg for 5 min to 10 min at room temperature Carefully aspirate and discard the medium leaving the pellet undisturbed

12 Calculate volume of cryopreservation medium based on the count performed at step 9 and resuspend pellet in cold cryopreservation medium at a viable cell density of 25 x 106 cellsmL (acceptable range 20 x 106 to 30 x 106) by gentle pipetting up and down

13 Dispense 1 mL of cell suspension using a 5 mL or 10 mL pipette into each 10 mL cryovial

14 Place filled cryovials at 2 oC to 8 oC until ready to cryopreserve A minimum equilibration time of 10 min but no longer than 45 min is necessary to allow DMSO to penetrate the cells

Note DMSO is toxic to the cells Long exposure in DMSO may affect viability

0 of 24


6 Cryopreservation of Cells

Material

Liquid nitrogen freezer

Cryomed Programmable freezer (Forma Scientific cat no 1010) or

Mr Frosty (Nalgene cat no 5100)

Isopropanol

Cryovial rack

a Cryopreservation using a rate-controlled programmable freezer

Method

A slow and reproducible cooling rate is very important to ensure good recovery of cultures A decrease of 1 degC per min to -80 degC followed by rapid freeze at about 15 degC to 30 degC per min drop to -150 degC will usually work for most animal cell cultures The best way to control the cooling process is to use a programmable rate-controlled electronic freezer unit Refer to the manufacturerrsquos handbook for detailed procedure

i Using the Cryomed

Starting the Cryopreservation Process

1 Check that the liquid nitrogen valve that supplies the Cryomed is open

2 Check the gauge to ensure that there is enough liquid nitrogen in the open tank to complete the freeze

3 Install the thermocouple probe so that the tip is immersed midway into the control fluid

Note Be sure that the thermocouple is centered in the vial and the vial is placed centered in the rack The probe should be changed after three uses or if it turns yellow to ensure accurate readings by the controller during the freezing process Old medium may have different freezing characteristics

4 Close and latch Cryomed door

5 Turn on microcomputer computer and monitor

6 Double click the ldquoCryomedrdquo icon The machine may need to be pre-programmed for specific cell type and medium

7 From the top of the screen select MENU RUN FUNCTIONS START RUN

Page 11 of 24


8 Fill out the box which appears on the screen Cell line ID TYPE OF SAMPLE MEDIA NUMBER OF SAMPLES

9 Hit the ESCAPE key and the Cryomed will cool to 4 C

10 Once Cryomed chamber has cooled to 4 C load cryovials onto racks and close the door

11 When the Cryomedrsquos chamber temperature and the sample temperature have reached approximately 4 C press the space bar to initiate the rate controlled cryopreservation process

Completing the Cryopreservation Process

1 When samples have reached -80C an alarm will sound To silence this select ALARM from the options at the top of the screen

2 Select MENU RUN FUNCTIONSrarr STOP Hit the ESCAPE key to return to the main menu and select EXIT

3 Immediately transfer vials to liquid nitrogen freezer

4 Shut down the microcomputer and then turn off the monitor

b Cryopreservation using ldquoMr Frostyrdquo

1 One day before freezing cells add 250 mL isopropanol to the bottom of the container and place at 2 oC to 8 oC

2 On the day of the freeze prepare cells for cryopreservation as described above

3 Insert cryovials with the cell suspension in appropriate slots in the container

4 Transfer the container to a -70 degC to -90 degC freezer and store overnight

5 Next day transfer cryovials to the vapor phase of liquid nitrogen freezer

Note Each container has 18 slots which can accommodate 18 cryovials in one freeze

Page 12 of 24


Important information when using the rate-controlled programmable freezer or a manual method (Mr Frosty) for cryopreservation of mammalian cells

Regardless which cooling method is used it is important that the transfer to the final storage location (between -130 degC and -96 degC) be done quickly and efficiently If the transfer cannot be done immediately the vials can be placed on dry ice for a short time This will avoid damage to cultures by inadvertent temporary warming during the transfer process Warming during this transfer process is a major cause of variation in culture viability upon thawing

Always keep the storage temperature below -130 degC for optimum survival Cells may survive storage at higher temperatures but viability will usually decrease over time The ideal storage container is a liquid nitrogen freezer where the cultures are stored in the vapor phase above the liquid nitrogen

Note ATCC does not have experience in the cryopreservation of the HCT 116 cells by any other method than the Cryomed programmable freezer

7 Storage

Store cryopreserved cells in the vapor phase of liquid nitrogen freezer (below -130 degC) for optimum long-term survival

Note Experiments on long-term storage of animal cell lines at different temperature levels

indicate that a -70 degC storage temperature is not adequate except for very short period of

time A -90 degC storage may be adequate for longer periods depending upon the cell line

preserved The efficiency of recovery however is not as great as when the cells are

stored in vapor phase of the liquid nitrogen freezer

Page 13 of 24


APPENDIX 1 PHOTOMICROGRAPHS OF NCI-PBCF-CCL247 (HCT 116)

CELLS

Figure 1 Photomicrograph of HCT 116 cells after one day post-freeze recovery Cells were plated at 10 x 105 viable cellscm2

Figure 2 Photomicrograph of HCT 116 cells after two days post-freeze recovery Cells were plated at 10 x 105 viable cellscm2

Page 14 of 24


40X 40X 40X

100X 100X 100X

500 microm500 microm500 microm

200 microm 200 microm 200 microm

Figure 3 Photomicrographs of HCT 116 cells at various time points after seeding at a cell density of 5 x 104 viable cellscm2

Page 15 of 24


APPENDIX 2 GROWTH PROFILE OF NCI-PBCF-CCL247 (HCT 116) CELLS

000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture

Figure 4 Growth curve for HCT 116 cells cells were plated at 5 x 104 viable cellscm2 population doubling time (PDT) is approximately 21 h

Page 16 of 24


APPENDIX 3 CYTOGENETIC ANALYSIS OF NCI-PBCF-CCL247 HCT 116 CELLS

NCI-PBCF-CCL247 Karyotype Results

Metaphase Spread Karyotype

Number of metaphase spreads counted 20

Band level 300-400

Number of metaphase spreads karyotyped 10

Chromosome range 45-46

Sex Male

Comments Aneuploid

Karyotype

46XYadd(10)(q26)add(16)(p133)add(18)(p112)[2] 45idem-Y[17] and 45idem

-16[1]

(ISCN nomenclature written based on a diploid karyotype)

Human diploid karyotype (2N) 46XX (female) or 46XY (male)

Page 17 of 24


Karyotype Summary

In the karyotype image arrows indicate regions of abnormality It should be noted that the karyotype description includes the observed abnormalities from all examined metaphase spreads but due to heterogeneity not all of the karyotyped cells will contain every abnormality

This is pseudo-diploid a human cell line of male origin containing 45 to 46 chromosomes per metaphase spread Structural abnormalities include rearrangements to chromosomes 10 16 and 18 Only about 10 of the examined cells contain a Y chromosome The lsquoidemrsquo designation indicates that the subclone(s) contain the same overall karyotype but with other changes For example the 45idem-Y clone described above has the same marker chromosomes as the clone 46XYadd(10)(q26) add(16)(p133)add(18)(p112) but is missing the

Y chromosome

The rearrangements include

Addition of unknown material to the short arms (designated by p) of chromosomes 16 and 18

Addition of unknown material to the long arm (designated by q) of chromosome 10

Numerical changes are based on a diploid karyotype which would contain two copies of

each chromosome (2N) Therefore a karyotype designation such as -16 indicates one copy

of structurally normal chromosome 16 and ndashY designates that no Y chromosome is present

(ISCN 2009 An International System for Human Cytogenetic Nomenclature (2009) Editors

Lisa G Shaffer Marilyn L Slovak Lynda J Campbell)

Karyotype Procedure

Cell Harvest Cells were allowed to grow to 80-90 confluence Mitotic division was

arrested by treating the cells with KaryoMaxreg colcemid for 20 minutes to 2 hours at 37degC

Cells were harvested using 005 Trypsin-EDTA treated with 0075M KCL hypotonic

solution and then fixed in three changes of a 31 ratio of methanolglacial acetic acid

Slide Preparation Slides were prepared by dropping the cell suspension onto wet glass

slides and allowing them to dry under controlled conditions

G-banding Slides were baked one hour at 90degC trypsinized using 10X trypsin-EDTA

and then stained with Leishmanrsquos stain

Microscopy Slides were scanned using a 10X objective and metaphase spreads were

analyzed using a 100X plan apochromat objective on an Olympus BX-41 microscope

Imaging and karyotyping were performed using Cytovisionreg software

Analysis Twenty metaphase cells were counted and analyzed and representative

metaphase cells were karyotyped depending on the complexity of the study

Page 18 of 24


Twenty metaphase cells were counted and analyzed and representative metaphase cells

were karyotyped depending on the complexity of the study

Summary of Karyotyping Procedure

G-band karyotyping analysis is performed using GTL banding technique G bands produced with trypsin and Leishman Slides prepared with metaphase spreads are treated with trypsin and stained with Leishmanrsquos This method produces a series of light and dark bands that allow for the positive identification of each chromosome

HCT 116 cell line karyotyping was carried out by Cell Line Genetics Inc (Madison WI 53719)

Page 19 of 24


APPENDIX 4 GLOSSARY OF TERMS

Confluent monolayer adherent cell culture in which all cells are in contact with other cells

all around their periphery and no available substrate is left uncovered

Split ratio the divisor of the dilution ration of a cell culture to subculture (eg one flask

divided into four or 100 mL up to 400 mL would be split ratio of 14)

Subculture (or passage) the transfer or transplantation of cells with or without dilution from one culture vessel to another

Passage No the total number of times the cells in the culture have been subcultured or passaged (with each subculture the passage number increases by 1)

Population doubling level (PDL) the total number of population doublings of a cell line since its initiation in vitro (with each subculture the population doubling increases in relationship to the split ratio at which the cells are plated) See Appendix 7

Population doubling time (doubling time) the time interval calculated during the logarithmic phase of growth in which cells double in number

Seeding density recommended number of cells per cm2 of substrate when inoculating a

new flask

Epithelial-like adherent cells of a polygonal shape with clear sharp boundaries between

them

Fibroblast-like adherent cells of a spindle or stellate shape

Page 20 of 24


APPENDIX 5 REFERENCE

1 Culture Of Animal Cells A Manual of Basic Technique by R Ian Freshney 6th

edition published by Wiley-Liss NY 2010

2 Schroy PC et al Detection of p21ras mutations in colorectal adenomas and

carcinomas by enzyme-linked immunosorbent assay Cancer 76 201-209 1995

PubMed 8625092

3 Brattain MG et al Heterogeneity of malignant cells from a human colonic carcinoma

Cancer Res 41 1751-1756 1981 PubMed 7214343

4 Sun L et al Autocrine transforming growth factor-beta 1 and beta 2 expression is

increased by cell crowding and quiescence in colon carcinoma cells Exp Cell Res

214 215-224 1994 PubMed 8082724

5 Santoro IM Groden J Alternative splicing of the APC gene and its association with

terminal differentiation Cancer Res 57 488-494 1997 PubMed 9012479

6 Brattain MG et al Enhancement of growth of human colon tumor cell lines by feeder

layers of murine fibroblasts J Natl Cancer Inst 69 767-771 1982 PubMed

6956756

7 Bender CM et al Inhibition of DNA methylation by 5-Aza-2-deoxycytidine

suppresses the growth of human tumor cell lines Cancer Res 58 95-101 1998

PubMed 9426064

8 Landers JE et al Translational enhancement of mdm2 oncogene expression in

human tumor cells containing a stabilized wild-type p53 protein Cancer Res 57

3562-3568 1997 PubMed 9270029

9 Kutchera W et al Protaglandin H synthase 2 is expressed abnormally in human

colon cancer evidence for a transcriptional effect Proc Natl Acad Sci USA 93

4816-4820 1996 PubMed 8643486

10 Wang R et al Cellular adherence elicits ligand-independent activation of the Met

cell-surface receptor Proc Natl Acad Sci USA 93 8425-8430 1996 PubMed

8710887

Page 21 of 24


APPENDIX 6 REAGENT LOT TRACEABILITY AND CELL EXPANSION TABLES

Table 4 Reagent Lot Traceability

Reagent Vendor Catalog Lot Expiration Date

Page 22 of 24

Table 5 Cell Expansion

FROM FLUID CHANGE

Observation under

microscope CELL COUNT TO

By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace


APPENDIX 7 CALCULATION OF POPULATION DOUBLING LEVEL (PDL)

Calculate the PDL of the current passage using the following equation

PDL = X + 3322 (log Y ndash log I)

Where X = initial PDL I = cell inoculum (number of cells plated in the flask) Y = final cell yield (number of cells at the end of the growth period)

APPENDIX 8 SAFETY PRECAUTIONS

Use at least approved Biological Safety Level 2 (BSL-2) facilities and procedures

Wear appropriate Personal Protective Equipment (PPE) such as isolation gown lab coat with sleeve protectors face shield and gloves

Use safety precautions when working with liquid nitrogen nitrogen vapor and cryogenically cooled fixtures

Use liquid nitrogen freezers and liquid nitrogen tanks only in areas with adequate ventilation Liquid nitrogen reduces the concentration of oxygen and can cause suffocation

Wear latex gloves over insulating gloves to prevent liquid nitrogen from soaking in and being held next to the skin Liquid nitrogen is extremely cold and will cause burns and frostbite Metal inventory racks tank components and liquid nitrogen transfer hoses exposed to liquid nitrogen or nitrogen vapor quickly cool to cryogenic temperatures and can cause burns and frostbite

Wear a full face mask when thawing and retrieving vials from liquid nitrogen freezer Danger to the technician derives mainly from the possibility that liquid nitrogen can penetrate the cryovial during storage On warming rapid evaporation of the nitrogen within the confines of such cryovial can cause an aerosol or explosion of the cryovial and contents

Page 24 of 24

Table of Contents

1 BACKGROUND INFORMATION ON HCT 116 CELL LINE 3

2 GENERAL INFORMATION FOR THE THAWING PROPAGATING AND CRYOPRESERVING OF NCI-PBCF- CCL247 (HCT 116) 3

3 REAGENTS 5

A PREPARATION OF COMPLETE GROWTH MEDIUM (MCCOYrsquoS 5A + 10 (VV) FBS) 5

4 THAWING AND PROPAGATION OF CELLS 6

A THAWING CELLS 6 B PROPAGATING CELLS 7 C SUBCULTURING CELLS 8

5 HARVESTING OF CELLS FOR CRYOPRESERVATION 9

6 CRYOPRESERVATION OF CELLS 11

A CRYOPRESERVATION USING A RATE-CONTROLLED PROGRAMMABLE FREEZER 11 i Using the Cryomed 11

B CRYOPRESERVATION USING ldquoMR FROSTYrdquo 12

7 STORAGE 13

APPENDIX 1 PHOTOMICROGRAPHS OF NCI-PBCF-CCL247 (HCT 116) CELLS 14

APPENDIX 2 GROWTH PROFILE OF NCI-PBCF-CCL247 (HCT 116) CELLS 16

APPENDIX 3 CYTOGENETIC ANALYSIS OF NCI-PBCF-CCL247 HCT 116 CELLS 17

APPENDIX 4 GLOSSARY OF TERMS 20

APPENDIX 5 REFERENCE 21

APPENDIX 6 REAGENT LOT TRACEABILITY AND CELL EXPANSION TABLES 22

APPENDIX 7 CALCULATION OF POPULATION DOUBLING LEVEL (PDL) 24

APPENDIX 8 SAFETY PRECAUTIONS 24






Biosafety Level 1




Source Organ colon







Culture Initiation



cellscm2



Complete growth

medium


(vv) FBS






oC to 8

oC when not in use

Cell Growth


oC



Special Growth

Requirements



2

Page 3 of 24


Subculture Medium







oC when not in

use

Subculture Method






viable cellscm 2

to5 x 104

viable cellscm 2


Viable

CellsmLCryovial



106


viable cellsmL)

Cryopreservation

Medium







Traceability of

materialreagents










Glossary of Terms

Safety Precaution




Page 4 of 24




125 cm2

(T-125) 3 mL to 6 mL

25 cm2

(T-25) 5 mL to 13 mL

75 cm2

(T-75) 10 mL to 38 mL

150 cm2

(T-150) 30 mL to 75 mL

175 cm2

(T-175) 35 mL to 88 mL

225 cm2

(T-225) 45 mL to 113 mL

3 Reagents












Serum (FBS)




magnesium chloride





(vv) FBS)




Page 5 of 24





Water bath




a Thawing cells

Method


35 oC to 37 oC










ethanol


Page 6 of 24


b Propagating cells

Method
















Page 7 of 24







DPBS





Method











Page 8 of 24

DPBS Rinse


125 cm2


25 cm2


75 cm2

(T-75) 4 mL to 15 mL 2 mL to 8 mL

150 cm2

(T-150) 8 mL to 30 mL 4 mL to 15 mL

175 cm2

(T-175) 9 mL to 35 mL 5 mL to 20 mL

225 cm2

(T-225) 10 mL to 45 mL 5 mL to 25 mL








DPBS





Sterile DMSO


Ice bucket with ice

Page 9 of 24


Method


number (c) date


















Page 10 of 24



Material




Isopropanol

Cryovial rack


Method


i Using the Cryomed










Page 11 of 24


















Page 12 of 24






7 Storage







Page 13 of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


0 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24






Biosafety Level 1




Source Organ colon







Culture Initiation



cellscm2



Complete growth

medium


(vv) FBS






oC to 8

oC when not in use

Cell Growth


oC



Special Growth

Requirements



2

of 24


Subculture Medium







oC when not in

use

Subculture Method






viable cellscm 2

to5 x 104

viable cellscm 2


Viable

CellsmLCryovial



106


viable cellsmL)

Cryopreservation

Medium







Traceability of

materialreagents










Glossary of Terms

Safety Precaution




Page 4 of 24




125 cm2

(T-125) 3 mL to 6 mL

25 cm2

(T-25) 5 mL to 13 mL

75 cm2

(T-75) 10 mL to 38 mL

150 cm2

(T-150) 30 mL to 75 mL

175 cm2

(T-175) 35 mL to 88 mL

225 cm2

(T-225) 45 mL to 113 mL

3 Reagents












Serum (FBS)




magnesium chloride





(vv) FBS)




Page 5 of 24





Water bath




a Thawing cells

Method


35 oC to 37 oC










ethanol


Page 6 of 24


b Propagating cells

Method
















Page 7 of 24







DPBS





Method











Page 8 of 24

DPBS Rinse


125 cm2


25 cm2


75 cm2

(T-75) 4 mL to 15 mL 2 mL to 8 mL

150 cm2

(T-150) 8 mL to 30 mL 4 mL to 15 mL

175 cm2

(T-175) 9 mL to 35 mL 5 mL to 20 mL

225 cm2

(T-225) 10 mL to 45 mL 5 mL to 25 mL








DPBS





Sterile DMSO


Ice bucket with ice

Page 9 of 24


Method


number (c) date


















Page 10 of 24



Material




Isopropanol

Cryovial rack


Method


i Using the Cryomed










Page 11 of 24


















Page 12 of 24






7 Storage







Page 13 of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24


Subculture Medium







oC when not in

use

Subculture Method






viable cellscm 2

to5 x 104

viable cellscm 2


Viable

CellsmLCryovial



106


viable cellsmL)

Cryopreservation

Medium







Traceability of

materialreagents










Glossary of Terms

Safety Precaution




of 24




125 cm2

(T-125) 3 mL to 6 mL

25 cm2

(T-25) 5 mL to 13 mL

75 cm2

(T-75) 10 mL to 38 mL

150 cm2

(T-150) 30 mL to 75 mL

175 cm2

(T-175) 35 mL to 88 mL

225 cm2

(T-225) 45 mL to 113 mL

3 Reagents












Serum (FBS)




magnesium chloride





(vv) FBS)




Page 5 of 24





Water bath




a Thawing cells

Method


35 oC to 37 oC










ethanol


Page 6 of 24


b Propagating cells

Method
















Page 7 of 24







DPBS





Method











Page 8 of 24

DPBS Rinse


125 cm2


25 cm2


75 cm2

(T-75) 4 mL to 15 mL 2 mL to 8 mL

150 cm2

(T-150) 8 mL to 30 mL 4 mL to 15 mL

175 cm2

(T-175) 9 mL to 35 mL 5 mL to 20 mL

225 cm2

(T-225) 10 mL to 45 mL 5 mL to 25 mL








DPBS





Sterile DMSO


Ice bucket with ice

Page 9 of 24


Method


number (c) date


















Page 10 of 24



Material




Isopropanol

Cryovial rack


Method


i Using the Cryomed










Page 11 of 24


















Page 12 of 24






7 Storage







Page 13 of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24




125 cm2

(T-125) 3 mL to 6 mL

25 cm2

(T-25) 5 mL to 13 mL

75 cm2

(T-75) 10 mL to 38 mL

150 cm2

(T-150) 30 mL to 75 mL

175 cm2

(T-175) 35 mL to 88 mL

225 cm2

(T-225) 45 mL to 113 mL

3 Reagents












Serum (FBS)




magnesium chloride





(vv) FBS)




of 24





Water bath




a Thawing cells

Method


35 oC to 37 oC










ethanol


Page 6 of 24


b Propagating cells

Method
















Page 7 of 24







DPBS





Method











Page 8 of 24

DPBS Rinse


125 cm2


25 cm2


75 cm2

(T-75) 4 mL to 15 mL 2 mL to 8 mL

150 cm2

(T-150) 8 mL to 30 mL 4 mL to 15 mL

175 cm2

(T-175) 9 mL to 35 mL 5 mL to 20 mL

225 cm2

(T-225) 10 mL to 45 mL 5 mL to 25 mL








DPBS





Sterile DMSO


Ice bucket with ice

Page 9 of 24


Method


number (c) date


















Page 10 of 24



Material




Isopropanol

Cryovial rack


Method


i Using the Cryomed










Page 11 of 24


















Page 12 of 24






7 Storage







Page 13 of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24





Water bath




a Thawing cells

Method


35 oC to 37 oC










ethanol


of 24


b Propagating cells

Method
















Page 7 of 24







DPBS





Method











Page 8 of 24

DPBS Rinse


125 cm2


25 cm2


75 cm2

(T-75) 4 mL to 15 mL 2 mL to 8 mL

150 cm2

(T-150) 8 mL to 30 mL 4 mL to 15 mL

175 cm2

(T-175) 9 mL to 35 mL 5 mL to 20 mL

225 cm2

(T-225) 10 mL to 45 mL 5 mL to 25 mL








DPBS





Sterile DMSO


Ice bucket with ice

Page 9 of 24


Method


number (c) date


















Page 10 of 24



Material




Isopropanol

Cryovial rack


Method


i Using the Cryomed










Page 11 of 24


















Page 12 of 24






7 Storage







Page 13 of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24


b Propagating cells

Method
















of 24







DPBS





Method











Page 8 of 24

DPBS Rinse


125 cm2


25 cm2


75 cm2

(T-75) 4 mL to 15 mL 2 mL to 8 mL

150 cm2

(T-150) 8 mL to 30 mL 4 mL to 15 mL

175 cm2

(T-175) 9 mL to 35 mL 5 mL to 20 mL

225 cm2

(T-225) 10 mL to 45 mL 5 mL to 25 mL








DPBS





Sterile DMSO


Ice bucket with ice

Page 9 of 24


Method


number (c) date


















Page 10 of 24



Material




Isopropanol

Cryovial rack


Method


i Using the Cryomed










Page 11 of 24


















Page 12 of 24






7 Storage







Page 13 of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24







DPBS





Method











of 24

DPBS Rinse


125 cm2


25 cm2


75 cm2

(T-75) 4 mL to 15 mL 2 mL to 8 mL

150 cm2

(T-150) 8 mL to 30 mL 4 mL to 15 mL

175 cm2

(T-175) 9 mL to 35 mL 5 mL to 20 mL

225 cm2

(T-225) 10 mL to 45 mL 5 mL to 25 mL








DPBS





Sterile DMSO


Ice bucket with ice

Page 9 of 24


Method


number (c) date


















Page 10 of 24



Material




Isopropanol

Cryovial rack


Method


i Using the Cryomed










Page 11 of 24


















Page 12 of 24






7 Storage







Page 13 of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24

DPBS Rinse


125 cm2


25 cm2


75 cm2

(T-75) 4 mL to 15 mL 2 mL to 8 mL

150 cm2

(T-150) 8 mL to 30 mL 4 mL to 15 mL

175 cm2

(T-175) 9 mL to 35 mL 5 mL to 20 mL

225 cm2

(T-225) 10 mL to 45 mL 5 mL to 25 mL








DPBS





Sterile DMSO


Ice bucket with ice

of 24


Method


number (c) date


















Page 10 of 24



Material




Isopropanol

Cryovial rack


Method


i Using the Cryomed










Page 11 of 24


















Page 12 of 24






7 Storage







Page 13 of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24


Method


number (c) date


















of 24



Material




Isopropanol

Cryovial rack


Method


i Using the Cryomed










Page 11 of 24


















Page 12 of 24






7 Storage







Page 13 of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24



Material




Isopropanol

Cryovial rack


Method


i Using the Cryomed










of 24


















Page 12 of 24






7 Storage







Page 13 of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24


















of 24






7 Storage







Page 13 of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24






7 Storage







of 24



CELLS



Page 14 of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24



CELLS



of 24


40X 40X 40X

100X 100X 100X




Page 15 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24


40X 40X 40X

100X 100X 100X




of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


Page 16 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24



000E+00

200E+05

400E+05

600E+05

800E+05

100E+06

120E+06

0 1 2 3 4 5 6

Via

ble

Cell

sc

m2

Days in Culture


of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



Page 17 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24






Band level 300-400



Sex Male

Comments Aneuploid

Karyotype


-16[1]



of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














Page 18 of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24


Karyotype Summary



Y chromosome









Karyotype Procedure














of 24







Page 19 of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24







of 24












new flask


them


Page 20 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24












new flask


them


of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

Page 21 of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24







PubMed 8625092





214 215-224 1994 PubMed 8082724





6956756



PubMed 9426064



3562-3568 1997 PubMed 9270029



4816-4820 1996 PubMed 8643486



8710887

of 24





Page 22 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24





of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24


FROM FLUID CHANGE

Observation under


By Date Flask qty

size

Pass Confluence

Add Replace

Volume

(in mL)

Viable cellsmL

Total viable cells

Viability

Split Ratio

Flask qty

size

Pass

PDL

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace

Add

Replace













Page 24 of 24













of 24

nci-pbcf-ccl247 (hct 116) colorectal carcinoma … · nci-pbcf-ccl247 (hct 116) colorectal...

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